CN1114694C - Procedures and materials for conferring disease resistance in plants - Google Patents

Abstract

Nucleic acids provided by the invention can encode polypeptides that confer resistance to Xanthomonas. The nucleic acid can be used to generate transgenic plants that are resistant to pathogens.

Description

Methods and materials for imparting disease resistance to plants

This is a continuation of co-pending US Patent Application No. 08/567,375 filed December 4, 1995, which is a continuation of US Provisional Application No. 60/004,645. It is also a continuation application of pending U.S. Patent Application No. 08/475,891 filed on June 7, 1995, which is a pending U.S. Patent Application No. Renewal application for .08/373,374. These applications are incorporated herein by reference.

field of invention

The present invention relates generally to plant molecular biology. In particular, it relates to nucleic acids and methods for conferring disease resistance in plants.

Statement of Rights in Inventions Made Under Government Sponsored Research and Development

This invention was made with government support under Lot No. GM47907 from the National Institutes of Health and Lot No. 9300834 from the United States Department of Agriculture. The government has certain rights in the invention.

Background of the invention

Gene loci that confer disease resistance have been identified in many plant species. Genetic analysis of many plant-pathogen interactions has shown that plants contain loci that confer resistance against specific species of pathogens containing complementary avirulence genes. Molecular characterization of these genes may provide a means of conferring disease resistance in a variety of crops.

Those plant resistance genes that have been characterized at the molecular level fall into 4 categories. One gene, Hml in maize, encodes a reductase enzyme effective against the fungal pathogen Cochliobolus Carbonum (Johal et al., Science , 258:985-987 (1992)). In tomato, the Pto gene confers resistance to Pseudomonas syringae expressing the avrPto avirulence gene (Martin et al., Science , 262:1432 (1993)). The predicted Pto gene encodes a serine threonine protein kinase. The tomato Cf-9 gene confers resistance to the fungus Cladosporium fulvum species carrying the avirulent gene Avr9 (Jones et al., Science , 266:789-793 (1994)). The tomato Cf-9 gene encodes a putative extracellular LRR protein. Finally, the RPS2 gene of Arabidopsis thaliana confers resistance to P. syringae expressing the avrRpt2 avirulence gene (Bent et al., Science , 265:1856-1860 (1994)). RPs2 encodes a protein with an LRR motif and a P-loop motif.

Bacterial wilts caused by Xanthomonas spp. affect nearly all agricultural crops and cause widespread crop losses worldwide. Oryza sativa bacterial wilt caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of this crop. Xoo varieties that induce resistance or susceptibility responses in rice cultivars with a distinctive resistance (Xa) gene have been identified. A source of resistance (Xa21) has been identified in the wild species of Oryza longistaminata (Khush et al., Proceedings of the International Workshop on Bacterial Blight of Rice (International Rice Research Institute, 1989) and Ikeda et al., Jpn J . Breed 40 (Suppl. 1): 280-281 (1990)). Xa21 is a dominant resistance locus that confers resistance to all known Xoo isolates and is uniquely characterized as the Xa gene that carries resistance to Xoo 6. Genetic and physical analyzes of the Xa21 locus have identified a number of closely linked markers on chromosome 11 (Ronald et al., Mol. Gen. Genet . 236:113-120 (1992)). However, the molecular mechanism by which Xa21 confers resistance to this pathogen is unclear.

Considerable effort has been devoted to cloning plant genes that confer resistance to various bacterial, fungal and viral diseases. Only one pest resistance gene has been cloned in monocots. Because monocot crops support the vast majority of people and animals in the world, the identification of disease resistance genes in these plants is of particular importance. The present invention addresses these and other needs.

Summary of the invention

The present invention provides an isolated nucleic acid construct comprising a RRK polynucleotide sequence which hybridizes to SEQ.ID.No.1 or SEQ.ID.No.3 under stringent conditions. A representative RRK polynucleotide sequence is the Xa21 sequence encoding the Xa21 polypeptide shown in SEQ.ID.No.4. RRK polynucleotides encode proteins with leucine-rich repeat motifs and/or cytoplasmic protein kinase domains. The nucleic acid constructs of the invention also contain a promoter operably linked to the RRK polynucleotide sequence. Promoters can be tissue-specific or constitutive.

The present invention also provides a nucleic acid construct comprising a promoter sequence from an RRK gene linked to a heterologous polynucleotide sequence. Representative heterologous polynucleotide sequences include structural genes that confer resistance to pathogens in plants.

The present invention also provides a transgenic plant containing a recombinant expression cassette containing a promoter from the RRK gene operably linked to a polynucleotide sequence; and a transgenic plant containing a recombinant expression cassette , the expression cassette contains a plant promoter operably linked to the RRK polynucleotide sequence. Although any plant can be used in the present invention, rice and tomato are conveniently used.

The invention also provides a method for enhancing the resistance of plants to Xanthomonas. The method comprises: introducing into a plant a recombinant expression cassette comprising a plant promoter operably linked to the RRK polynucleotide sequence. This method can be conveniently applied to rice and tomato plants.

definition

The term "plant" includes whole plants, plant organs (such as leaves, stems, roots, etc.), seeds and plant cells and their progeny. The class of plants that can be used in the methods of the invention generally extends as wide as the types of higher plants amenable to transformation techniques, including monocots and dicots.

A "heterologous sequence" is a sequence from a different species or, if from the same species, substantially modified from its original form. For example, a promoter operably linked to a heterologous structural gene may be from a species different from that from which the structural gene was derived, or, if from the same species, either or both are sufficiently regulated for their original form. grooming.

"RRK genes" are members of a novel class of disease resistance genes encoding RRK polypeptides containing an extracellular LRR domain, a transmembrane domain, and a cytoplasmic protein kinase domain (as described in, e.g., RLK5, Pto, and Fen (Martin et al., The Plant Cell 6: 1543-1552 (1994)). As used herein, the LRR domain is a repeating unit of about 24 residues as shown in Figure 1 and found in Cf-9 and RLK5 area.). Using the sequences disclosed herein and standard nucleic acid hybridization and/or amplification techniques, one of skill can identify members of such genes. For example, a nucleic acid probe from the Xa21 gene can detect a polymorphism segregated with the blast (Pyricularia oryzae) resistance gene (Pi7) in 58 recombinant inbred lines of rice. The same probes also detect polymorphisms in near-isogenic lines carrying the xa5 and Xa10 resistance genes.

In certain preferred embodiments, members of such disease resistance genes can be identified by their ability to be amplified by degenerate PCR primers corresponding to the LRR and kinase domains. For example, primers have been used to isolate homologous genes in tomato. Representative primers for this purpose are tcaag caaca atttg tcagg nca a/gat a/c/t cc (for the LRR domain sequence GQIP) and taaca gcaca ttgct tgatt tnan g/a tcncg g/atg (for the kinase domain sequence HCDIK).

"Xa21 polynucleotide sequence" is a subsequence or full-length polynucleotide sequence of a Xa21 gene, such as a rice Xa21 gene, which, when present in a transgenic plant, will target Xanthomonas (such as X. oryzae ) resistance to the plant. Representative polynucleotides of the invention include the coding region of SEQ.ID.No.3. Xa21 polynucleotides are typically at least about 3100-6500 nucleotides in length, usually about 4000-4500 nucleotides in length.

"Xa21 polypeptide" is the gene product of the Xa21 polynucleotide sequence, which has the activity of Xa21, that is, it can confer resistance to Xanthomonas. Like other RRK polypeptides, the Xa21 polypeptide is characterized by the presence of an extracellular domain containing leucine rich repeats (LRR) and/or a cytoplasmic protein kinase domain. A representative Xa21 polypeptide of the invention is included in SEQ.ID.No.4.

In the expression of a transgene, one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be "substantially identical" to the gene sequence of origin. These variants are specifically encompassed by this term as described below.

In cases where the inserted polynucleotide sequence is transcribed and translated to produce a functional RRK polypeptide, the skilled artisan will recognize that, because of codon degeneracy, there are a large number of polynucleotide sequences that can encode the same polypeptide . These variants are specifically covered by the term "RRK polynucleotide sequence". In addition, the term specifically includes a full-length sequence that is substantially identical to the RRK gene sequence (the determination method is described below), as well as a sequence that encodes a protein that retains the function of the RRK protein. Thus, in the context of the rice RRK gene disclosed herein, the above term includes variant polynucleotide sequences that are substantially identical to the sequences disclosed herein and that encode proteins capable of conferring a gene in a transgenic plant containing the sequence. Resistance against Xanthomonas or other plant diseases or pests.

Two polynucleotides or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is identical when aligned for maximum correspondence as described below. The term "complementary to" is used herein to mean that the complementary sequence is identical to a referenced polynucleotide sequence in whole or in part.

Sequence comparisons between two (or more) polynucleotides or polypeptides are generally performed by comparing the order of the two sequences over a segment or "comparison window" to identify or compare local regions of sequence similarity. The fragments used for comparison purposes are at least about 20 contiguous positions, usually about 50-200, more usually about 100-150, wherein after the two sequences are optimally aligned, the sequence is compared with only The same number of consecutive positions are compared to the reference sequence.

For comparison, optimal alignment of sequences can be performed using the local homology algorithm of Smith and Waterman Adv. Appl. Math . 2: 482 (1981), Needleman and Wunsch Journal of Molecular Biology (J. Mol. Biol.) 48: 443 (1970), the similarity search method of Person and Lipman Proc. Natl . Acad. Sci . (USA) 85: 2444 (1988), or These algorithms were performed by computer (GAP, BESTFIT, FASTA, and TFASTA, in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection.

"Percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence within the comparison window may include the sequence with which the two sequences were optimally aligned for optimal alignment. (it does not contain insertions or deletions) when compared to additions or deletions (such as gaps). The percentage is calculated by determining the number of identical nucleic acid base or amino acid residue positions in the two sequences, dividing the number of matching positions by the total number of positions within the comparison window, and then multiplying the result by 100 to obtain the percent sequence identity .

The term "substantially identical" to a polynucleotide means that the polynucleotide sequence contains at least 60% sequence identity, preferably at least 80%, when compared with a reference sequence using standard parameters and the above-mentioned programs (preferably BESTFIT). % sequence identity, more preferably at least 90% sequence identity, optimally at least 95% sequence identity. Those skilled in the art will recognize that appropriate adjustments to these values can be made to determine the protein encoded by the two nucleotide sequences by taking into account factors such as codon degeneracy, amino acid similarity, reading frame position, etc. the sameness. For these purposes, substantial amino acid sequence identity generally means at least 40%, preferably at least 60%, more preferably at least 90%, and most preferably at least 95% sequence identity. Polypeptides that are "substantially similar" share sequences as described above, except that residue positions that are not identical may be replaced by conservative amino acid changes. Conservative amino acid substitutions refer to the substitution of residues with similar side chains for each other. For example, a group of amino acids with aliphatic side chains are glycine, alanine, valine, leucine, isoleucine; a group of amino acids with aliphatic hydroxyl side chains are serine, threonine; The group of amino acids with side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, tryptophan; the group of amino acids with basic side chains is lysine acid, arginine, and histidine; and a group of amino acids with sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine - Glutamine.

Another indication that the nucleotide sequences are substantially identical is that the two molecules hybridize to each other under suitable conditions. Suitable conditions may be of high or low stringency, which will vary in different cases. Generally, stringent conditions are selected to be about 5-20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under a defined ionic strength and pH) at which approximately 50% of the target sequence hybridizes to a perfectly matched probe. Generally, stringent wash conditions are at pH 7, a salt concentration of about 0.02 molar and a temperature of at least about 60°C. However, nucleic acids that do not hybridize to each other under stringent conditions may still be substantially identical if the polypeptides they encode are substantially identical. This occurs, for example, when a copy of the nucleic acid is created with the maximum degree of codon degeneracy permitted by the genetic code. For Southern hybridization, high stringency wash conditions may include at least one wash in 0.1 x SSC at 65°C.

Nucleic acids of the invention can be identified from cDNA or genomic libraries using standard procedures and using a nucleic acid disclosed herein (eg, SEQ. ID. No. 1 or 3) as a probe. Low stringency hybridization conditions typically include at least one wash with 2 x SSC at 65°C. Washing is preferably followed by washing with 1×SSC at 65°C.

As used herein, a homologue of a particular RRK gene, such as the rice Xa21 gene disclosed herein, is a second encoded amino acid sequence that is at least 25% identical or 45% similar (as determined above) to the polypeptide sequence of the first gene product Genes for proteins (in the same species or in different species). It is generally believed that homologues share a common evolutionary origin.

Brief description of the drawings

Figure 1 is a comparison of proteins with leucine-rich repeats.

Figures 2A-F show partial restriction maps of BAC and cosmid clones containing regions hybridizable to Xa21-specific probes.

Figure 3 shows the restriction map of pB822 (the most active copy).

Figure 4 shows the results of a test measuring Xanthomonas resistance in transgenic plants containing the Xa21 gene from the pB822 clone.

Figure 5 shows the location map of TRK1.

Figure 6 shows the location map of TRL1.

Description of the preferred embodiment

The present invention relates to plant RRK genes, such as rice Xa21 gene. Nucleic acid sequences from the RRK genes, especially the Xa21 gene, can be used to confer resistance to Xanthomonas or other pathogens in plants. The present invention can be used to confer resistance in all higher plants susceptible to pathogen infection. Thus, the present invention is applicable to a large and varied range of plant types, including species from the following genera: Juglans, Fragaria, Lotus, Medicago, Bean ( Onobrychis, Trigonella, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot , Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Mann Datrua, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Chicory Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panicum (Panieum), Pennisetum, Ranunculus, Senecio, (Salpiglossis), Cucumis, Browaalia, Glycine, Pisum, Phaseolus Phaseolus, Lolium, Zea, Avena, Hordeum, Secale, Triticum and Sorghum .

The following Example section describing the isolation and characterization of the Xa21 gene in rice is representative of a general method for isolating the Xa21 gene and other RRK genes. The isolated gene can then be used to construct recombinant vectors to confer RRK gene expression in transgenic plants.

In general, the nomenclature and laboratory procedures in recombinant DNA techniques described below are those well known and commonly used in the art. Standard techniques are used for cloning, isolation of DNA and RNA, amplification and purification. Generally, enzymatic reactions involving DNA ligase, DNA polymerase, restriction enzymes, etc. are performed according to the manufacturer's instructions. These and various other techniques are generally performed in accordance with Sambrook et al., "Molecular Cloning - A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989).

Isolation of Xa21 and related RRK genes can be accomplished using a variety of techniques. For example, oligonucleotide probes based on the sequences disclosed herein can be used to identify desired genes in cDNA or genomic DNA libraries. For the construction of genomic libraries, large fragments of genomic DNA can be generated by random fragmentation (eg, by restriction enzymes) and then ligated with vector DNA to form concatemers that can be packaged into a suitable vector. To prepare a cDNA library, mRNA is isolated from a desired organ such as a leaf and then used to prepare a cDNA library containing RRK gene transcripts. Alternatively, cDNA can be prepared from mRNA extracted from other tissues expressing the RRK gene or homologue.

The cDNA or genomic libraries are then screened with probes based on cloned RRK genes, such as the rice Xa21 gene disclosed herein. Probes can be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species.

Alternatively, amplification techniques can be used to amplify a nucleic acid of interest from a nucleic acid sample. For example, the sequence of RRK and related genes can be directly amplified from genomic DNA, cDNA, genomic library or cDNA library by polymerase chain reaction (PCR) technology. PCR and other in vitro amplification methods can also be used, for example, to clone nucleic acid sequences encoding proteins to be expressed, to prepare nucleic acids to be used as probes for the presence or absence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes.

Suitable primers and probes for identifying RRK sequences from tissues can be derived by comparing the sequences provided herein. For a general review of PCR, see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J., and White, T. eds.), Academic Press, San Diego (1990), in It is incorporated herein by reference.

Polynucleotides can also be synthesized using well-known techniques, such as those described in the scientific literature. See, eg, Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982) and Adams et al., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments can then be obtained either by synthesizing complementary strands, annealing the strands together under appropriate conditions, or by adding complementary strands by DNA polymerase with appropriate primer sequences.

Isolated sequences prepared as described herein can then be used to provide RRK gene expression in desired plants, thereby providing Xanthomonas resistance. The skilled artisan will recognize that a nucleic acid encoding a functional RRK protein (eg, SEQ. ID. No. 2 and 4) need not have the same sequence as the representative genes disclosed herein. Furthermore, like other proteins, the polypeptides encoded by RRK genes have different domains that perform different functions. Therefore, the RRK gene sequence does not have to be full-length, as long as the desired protein functional domains are expressed. As described in detail below, the proteins of the invention contain an extracellular leucine-rich repeat domain, and an intracellular kinase domain. Modified protein chains can be conveniently designed using a variety of recombinant DNA techniques well known to those skilled in the art. For example, chains can be made to differ from the naturally occurring sequence at the level of primary structure by amino acid substitutions, insertions, deletions, and the like. Modifications may also include exchanging domains from the proteins of the invention with related domains from other pest resistance genes. For example, the extracellular domain of the protein of the invention, including the region of the leucine-rich repeat, can be replaced by the extracellular domain of the tomato Cf-9 gene, thereby conferring resistance to rice fungal pathogens. These modified forms can be used in numerous combinations to produce the final modified protein chain.

To use the isolated RRK sequences in the techniques described above, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a variety of higher plant species are well known and described in the technical and scientific literature. See, eg, Weising et al., Ann. Rev. Genet. 22:421-477 (1988).

A DNA sequence encoding a desired RRK polypeptide, eg, a cDNA or a genomic sequence encoding a full-length protein, can be used to construct a recombinant expression cassette that can be introduced into a desired plant. An expression cassette typically includes a RRK polynucleotide operably linked to transcriptional and translational initiation regulatory sequences that direct the transcription of the RRK gene sequence in the desired tissue of the transformed plant.

For example, plant promoter fragments that direct expression of RRK in all tissues of regenerated plants can be used. Such promoters, referred to herein as "constitutive" promoters, are active under most environmental conditions and stages of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1' or 2' promoter from the T-DNA of Agrobacterium tumafaciens, and the promoters known to the skilled person from various Other transcription initiation regions of different plant genes.

Alternatively, a plant promoter can direct the expression of the RRK gene in a specific tissue, or under more precise environmental or developmental control. Such promoters are referred to herein as "inducible" promoters. Environmental conditions that can affect transcription from an inducible promoter include pathogen attack, anaerobic conditions, or the presence of light.

Examples of promoters that are under developmental control include promoters that initiate transcription only in certain tissues such as leaves, roots, fruit, seeds or flowers. A promoter may also operate differently, depending on its location in the genome. Thus, an inducible promoter is fully or partially constitutive at certain positions.

The endogenous promoter from the RRK gene of the present invention can be used to direct the expression of the gene. These promoters can also be used to direct the expression of heterologous structural genes. Thus, a promoter can be used in a recombinant expression cassette to drive the expression of a gene that confers resistance to any pathogen including fungi, bacteria, and the like.

To identify promoters, the 5' portions of the clones described here were analyzed for sequence signatures of promoter sequences. For example, promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually located 20-30 base pairs upstream from the transcription start site. In plants, further upstream of the TATA box, at positions -80 to -100, there is typically a promoter element with a series of adenines surrounding the trinucleotide G (or T)NG. J. Messing et al., Genetic Engineering in Plants, pp. 221-227 (eds. Kosage, Meredith and Hollaender, 1983).

If proper polypeptide expression is desired, a polyadenylation region should be introduced 3' to the RRK coding region. The polyadenylation region may be from a native gene, or from a variety of other plant genes, or from T-DNA.

Vectors containing sequences from RRK genes typically contain marker genes that confer a selectable phenotype on plant cells. For example, marker genes may encode antibiotic resistance, especially resistance to antibiotics such as kanamycin, G418, bleomycin, hygromycin, resistance to herbicides such as resistance to chlorosluforon or Basta .

These DNA constructs can be introduced into the genome of the desired plant host using a variety of conventional techniques. For example, using techniques such as electroporation, PEG perforation, particle bombardment, and microinjection on plant cell protoplasts or embryo callus, DNA constructs can be directly introduced into the genomic DNA of plant cells, or by impact methods such as DNA particle bombardment, DNA constructs can be introduced directly into plant tissue. Alternatively, the DNA construct can be combined with appropriate T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence function of the Agrobacterium tumefaciens host can direct the insertion of the construct and adjacent markers into the plant cell DNA when the cells are infected by the bacteria.

Transformation techniques are well known in the art and fully described in the scientific and patent literature. Methods for introducing DNA constructs using polyethylene glycol are described in Paszkowski et al., Embo J. 2717-2722 (1984). Electroporation techniques are described in Fromm et al., PNAS 82:5824 (1985). The bombardment transformation technique is described in Klein et al., Nature , 327:70-73 (1987). Cereal species such as rye (de la Pena et al., Nature , 325:274-276 (1987)), maize (Rhodes et al., Science , 240:204-207 (1988)) and rice ( Shimamoto et al., Nature , 338:274-276 (1989), by electroporation; Li et al., Plant Cell Rep. 12:250-255 (1993), by bombardment).

A. tumefaciens-mediated transformation techniques are well described in the scientific literature. See, eg, Horsch et al., Science , 233:496-498 (1984) and Fraley et al., Proc. Natl. Acad. Sci. , 80:4803 (1983). Although Agrobacterium is mainly used on dicotyledonous plants, some monocotyledonous plants can also be transformed with Agrobacterium. Transformation of rice with Agrobacterium is described, for example, in Hiei et al., Plant J. 6:271-282 (1994).

Transformed plant cells derived using any of the transformation techniques described above can be cultured to regenerate whole plants having the transformed genotype and thus the desired RRK-controlled phenotype. This regenerative technique relies on the action of certain plant hormones in the tissue culture growth medium, typically an antibiotic and/or herbicide marker that has been included with the RRK nucleotide sequence. Regeneration of plants from cultured protoplasts is described in Evan et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture , pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. .21-73, CRC Press, Boca Raton, 1985. Regeneration can also be from plant callus, explants, organs or parts thereof. This regeneration technique is fully described in Klee et al., Ann. Rev. Of Plant Phys. 38:467-486 (1987).

The methods of the invention are particularly useful for introducing RRK polynucleotides into transformed plants in a manner and under conditions not found in nature. In particular, RRK polynucleotides may be expressed frequently or in large quantities that are different from the characteristics of native plants.

The skilled artisan also recognizes that after the expression cassette has been stably introduced into a transgenic plant and proven operable, it can be introduced into other plants by sexual crossing. Depending on the species to be crossed, any of a number of standard hybridization techniques can be used.

The effect of modification of RRK gene expression can be determined by detecting an increase or decrease at the mRNA level, for example by Northern blotting. In addition, phenotypic effects of gene expression can be detected by measuring lesion length in plants. Suitable assays for determining resistance are described below.

The following examples are given by way of illustration and not by way of limitation.

Example 1

Map-based cloning can also be used to isolate plant genes. The method involves the identification of DNA markers that are closely linked to the gene of interest. A requirement for successful map-based cloning and physical analysis of large chromosomal regions is the availability of gene libraries containing large inserts of genomic DNA. Recently, Shizuya.H. et al. described a bacterial artificial chromosome (BAC) system in Proc. fragment. The system uses F-factor-based vectors capable of containing human genomic DNA fragments larger than 300kb. DNA can be efficiently cloned in Escherichia coli (E.coli.), easy to operate and stable in storage. The following is a description of the isolation of the gene of the invention using this technique.

Isolation of BAC and cosmid clones carrying Xa21-related sequences

BAC Cloning A. Materials and Methods Preparation of Rice High Molecular Weight DNA

A rice line from the International Rice Research Institute (IRRI), IR-BB21 carrying Xa-21, was used as plant material. Plants are grown for 3 to 5 weeks in the greenhouse. Leaf tissues were collected, washed with distilled water, and crushed. According to Hatano, S. et al. (Plant Science Journal), 83, 55-64 (1992) and Zhang, HB et al. in Plant (Plant), 7, 175-184 (1994), carry out High molecular weight DNA was extracted from rice tissue with the following modification: In liquid nitrogen, approximately 20 g of leaf tissue was ground into powder with a cold mortar and pestle. Suspend the powder by stirring in 200 ml of cold nuclear extraction (NE) buffer (1MM spermidine, 1mM spermine, 10mM _Na2EDTA , 10mm Trizma base, 80mM KCl, 0.15% Triton-X100 and 0.4M sucrose, pH 9.4 )middle. This mixture was filtered through two layers of cheese cloth into GSA bottles and centrifuged at 1200 g for 20 minutes at 4°C. Discard the supernatant and resuspend the nuclear pellet (light green) in 50 ml of cold NE buffer. The resuspended pellet was filtered through an 80-micron sieve into a 50-ml tube to remove green tissue debris, and centrifuged at 1000g for 10 minutes. The pellet was resuspended and centrifuged as above, but not filtered through an 80 micron sieve. The cell nucleus pellet (approximately 5× ¹⁰⁸ /ml) was resuspended in 2.5ml SCE buffer (1M sorbitol, 0.1M sodium citrate, 60mM EDTA, pH7.0), and embedded in 2.5ml 1% low melting point (LMP ) in agarose (Ultrapure). Blocks of 80 μl were incubated in 25 ml of ESP solution (0.5 M EDTA, pH 9.3, 1% sodium lauryl sarcosine, 5 mg/ml proteinase K, Boehringer Mannheim) at 50° C. for 2 days with a buffer change during the period. Each block contained 5 μg DNA. Partial digestion of high molecular weight DNA and size separation using PFGE

The agarose block was dialyzed twice for 1 hour against TE (10 mm Tris-HCl and 1 mM EDTA, pH 8.0) supplemented with 1 mm PMSF (phenylmethylsulfonyl fluoride) at 50 °C, then buffered with HindIII at room temperature solution (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ and 1 mM dithioerythritol, pH 7.9) were equilibrated twice for 1 hour. The blocks were melted at 65°C for 15 minutes, incubated at 37°C for 5 minutes, and then partially digested. Add 5 to 7 units/block of HindIII (NEB, USA) to the DNA solution, and incubate at 37°C for 30 minutes. The reaction was stopped by adding 1/10 volume of 0.5% EDTA pH 8.0. The partially digested DNA was immediately added to 0.8% LMP agarose with a tip pipette cut to an inner diameter of 2 mm, and separated by PFGE (CHEF DR II system, BioRad USA). Libraries were constructed using two different PFGE methods. In the first, the gel was run at 150 V, using a start and stop switching time of 8 seconds, for 16 hours at 14°C. Unseparated DNA (≥200kb) was concentrated into a narrow band. The second, gel electrophoresis at 150V, switching time increased from 60 seconds to 90 seconds, at 14°C for 16 hours. Gels containing partially digested DNA from both methods were excised and immersed in TE while the marker lanes of the gel were stained with ethidium bromide. Agarose strips containing fragments larger than 200 kb (first PFGE method) or 250-350 kb fragments (second method) were excised from the gel. The agarose strips were equilibrated in TE for 2 hours at 4°C, put into a 1.5ml test tube, melted at 60°C for 10 minutes, and gelatinized (Gelase) (Epicenter, USA) (1 unit of enzyme per 100g of agarose) Digest and incubate at 45°C for 1 hr. This DNA solution was used directly in the ligation reaction. Vector preparation and isolation, and ligation reactions

The vector pBeloBAC II was provided by Drs. H.Shizuya and M.Simon (California Institute of Technology, USA). This vector has the lacz gene inserted in pBAC108L. Shizuya et al. (1992). Inoculate a single colony into 5 ml of LB medium containing 12.5 μg/ml chloramphenicol, grow at 37°C for 4 to 5 hours, and then add 6 ml of LB medium. The inoculum was grown at 37°C for approximately 16 hours until an _OD600nm of 1.3-1.5 was reached. Plasmids were isolated using Qiagen's Plasmid Max Isolation Kit (Qiagen, USA). Vector DNA was purified by cesium chloride/ethidium bromide equilibrium centrifugation at 45,000 RPM for 60 hours. Using a fixed angle rotor 70.1 (Beckman, USA), the rotor was slowed down to 35,000 RPM and centrifuged for 1 hour to loosen the gradient. The plasmid was digested completely with HindIII and analyzed by gel electrophoresis. The vector ends were dephosphorylated with HK phosphatase (Epicenter, USA) at 30°C for 1 hour, using 1 unit of enzyme per 1 μg of vector DNA. HK phosphatase was inactivated by heating at 65°C for 30 minutes. A ligation reaction was performed in 100 μl in which about 40 ng of size-selected rice DNA (about 85 μl) was ligated with 10 ng of HindIII-digested vector (1 μl) with 400 units of T4 DNA ligase (NEB, USA) (molar ratio about 10 1, carrier excess). Before the transformation, the ligation reaction solution was dialyzed against TE at 4° C. overnight in an ULTRAFREE-MC filter tube (Millipore, USA). BAC conversion

According to the following settings, use Cell-Porator (GIBCO-BRL, USA) to transform competent Escherichia coli DH10B cells (GIBCOBRL, USA) by electroporation: voltage: 400; charge speed: fast; pressurization resistance: 4,000; : 300μ; resistance: low. For each electroporation, mix 13 μl competent cells with 0.5-1.0 μl ligation reaction solution. After electroporation, cells were transferred to 1ml of SOC solution (2% Bacterial Tryptone, 0.5% Bacterial Yeast Extract, 10MM NaCl, 2.5mM KCl, 10mM MgCL ₂ , 10mM MgSO ₄ , 20mM Glucose, pH 7.0) , and incubated at 37° C. with gentle shaking (90-95 RPM) for 45 minutes. Disperse cells in LB culture plates containing chloramphenicol (12.5 μg/ml), X-gal (40 μg/ml) and IPTG (isopropylthio-β-D-galactoside) (0.072 μg/ml) . Plates were incubated at 37°C for 24 hours. The white colony containing the rice DNA insert was selected onto a fresh LB culture plate for a second color screening. The BAC clones were transferred to a 384-grid microtiter plate (Genetix, UK) containing 60 μl of LB freezing buffer (36 mM K ₂ HPO ₄ , 13.2 mM KH ₂ PO ₄ , 1.7 mM citric acid, 0.4MM MgSO ₄ , 6 .SmM (NH ₄ ) ₂ SO ₄ , 4.4% v/v glycerol, 12.5 μg/ml chloramphenicol, LB), cultured at 37°C for 24 hours. Since more than 95% of the colonies were still white in the second screening, only one screening was performed in the following experiments, and the white colonies were directly selected on the 384-grid microtiter plate. The library was replicated twice and stored in two different -80°C freezers. Membrane preparation

BAC colonies from each well of the 384-well microtiter plate were replicated onto Hybond N ⁺ filters (Amersham, USA). The filters were placed in plastic boxes containing LB/agarose and 12.5 μg/ml chloramphenicol and the boxes were incubated overnight at 37°C until colonies reached approximately 2 to 3 mm in diameter. Filters were treated as described by Nizetic, D et al. Nucl. Acids Res. 19, 182 (1990); Hoheisel, JD et al. Cell 73, 109-120 (1993). Hybridization and washing conditions were as described by Hoheisel et al. (1993). Probes are labeled using random primer extension reactions. Feinberg, AP and Vogelstein, B., Anal. Biochem. 132, 6-13 (1983); Appendices 137, 266-267 (1984). B. Results

The BAC library described above included 11,000 clones. The library was constructed in two ways. The first half of the library with 7269 clones was size-selected once using the compressed band method described in Ramsay, M and Wicking, C., in Protocols in Human Molecular Genetics, 197-221 (1991) constructed. The second half of the library with 3731 clones was constructed using double size selection on partially digested DNA. However, two size selections did not improve the average size of DNA inserts. Apparently, small molecules of DNA are still present in the size-selected DNA solution (only 250 to 350 kb of DNA was isolated). The following experiments demonstrate that double size selection of DNA between 350 and 500 kb for ligation increases the larger average insert size in BAC clones. Of the 54 random BAC clones selected from the library, 50 contained rice DNA (93.0%). Some clones contained no insert (7%). The size of the DNA insert ranged from 30 to 250 kb with an average of 125 kb.

The high molecular weight DNA used to construct the BAC library was isolated from purified rice nuclei. Low speed centrifugation (<1000 g) removed most of the chloroplasts and mitochondria. The low frequency (<0.3%) of finding chloroplast or mitochondrial clones in new BAC libraries reduces the likelihood of organelle DNA/nucleus DNA junctions.

The BAC library was used to construct a set of contiguous clones (contig) spanning the Xa21 locus. With two Xa21-linked DNA markers, RG103 (1 kb, see Ronald et al., Molecular Genetics (Mol. Gen. Genet.) 236:113-120 (1992)) and pTA818 (1.2 kb, equivalent to Ronald et al. RAPD818) to screen the BAC library. Eight copies of RG103 were found in the Xa21-containing line and hybridized with eight genomic HindIII DNA fragments in this line. These fragments are all genetically and physically linked to the Xa21 disease resistance locus. pTA818 hybridizes with 2 DNA fragments, at least one of which is linked to the Xa21 locus. Ronald et al. (1992).

7296 BAC clones were hybridized with pTA818 (2 copies) and RG103 (8 copies) probes. Seven BAC clones hybridizing to RG103 and 5 BAC clones hybridizing to pTA818 were identified, respectively. BAC DNA was isolated from these clones and digested with HindIII. DNA fragments were separated using PFGE. Southern analysis showed that 7 BAC clones hybridized with RG103 carried 4 different copies of HindIII fragment of RG103 genome. The probe hybridizes to a 4.3kb and 9.5kb fragment and to a 9.6kb and 6.2kb fragment. The sizes of DNA fragments were deduced from lambda DNA digested with HindIII.

Four BAC clones carrying one pTA818 HindIII fragment were isolated and one BAC clone carrying the other copy was identified. Hybridization of BAC-containing pTA818 to marker PTA24g (equivalent to RAPD248 of Ronald et al. (1992)) confirmed that the two cloned RAPD marker genes were both within 60 kb and contained each other. Ronald et al. (1992).

Twelve BAC clones hybridizing to 2 cloned DNA sequences (corresponding to 10 DNA fragments in the rice genome) were identified, which was slightly lower than screening based on 2x genome equivalents (7296 colonies, 450,000 kb genome, average insert size 125 kb) Expected 20 colonies. Specifically, the pTA818 sequence and the 4 RG103 hybrids (4 out of 8) were overrepresented in this part of the library. In contrast, the other 4 RG103 sequences were underrepresented. The DNA insert size of these clones ranged from 40 to 140 kb.

Cosmid Cloning A. Materials and Methods Preparation of high molecular weight (HMW) DNA from rice leaves

Rice line 1188 carrying the Xa21 locus was used as plant material for isolation of HMW DNA. 120 g of 4- to 6-week-old leaf tissue were collected and ground to a fine powder in liquid nitrogen with a cold mortar and pestle. Then stir and suspend the powder in 800ml of cold H buffer [4mM spermidine, 1mM spermine, 10mM EDTA, 10mM Tris-HCl, 80mM KCl, 0.5M sucrose, 1mM PMSF (phenylmethylsulfonyl chloride, add before use) ), 0.5% (v/v) Triton-X100, 1/1000 (v/v) β-mercaptoethanol (added before use), pH9.5]. The mixture was filtered through an 80 micron sieve into a GSA bottle, the pellet was resuspended in 400 ml H buffer and filtered again. The two filtrates were combined and centrifuged at 3500 rpm for 10 minutes at 4°C. The pellet was resuspended in 300 ml wash buffer (same as H buffer but without PMSF and β-mercaptoethanol) and centrifuged at 3500 rpm for 10 minutes at 4°C. The pellet was washed two more times until the color of the pellet was light green. Resuspend the pellet in 40ml washing buffer, add an equal volume of lysis buffer (2% sodium lauryl sarcosine, 100mM Tris-HCl, 0.5M EDTA, pH 9.5) lyses nuclei. Reaction was carried out at 50° C. for 5 hours, followed by extraction with an equal volume of phenol-chloroform-isoamyl alcohol (24:24:1) for 30 minutes at room temperature (gentle inversion), thereby removing protein. HMW DNA was precipitated by gentle addition of a layer of 1/10 volume of 3M sodium acetate (pH 5.5) and a layer of 2 volumes of ethanol and gentle inversion several times. Finally, the DNA was aspirated from ethanol with a wide-bore pointed pipette, washed with 70% ethanol, dried, dissolved in 1ml TE (10mM Tris-HCl, 1mM EDTA, pH 8.0) and allowed to stand overnight. Typically, 250 μg of HMW DNA can be isolated from 120 g of leaves. Preparation of insert DNA (A) Partial digestion of HMW DNA

pre-test. Mix 30 μg (70 μl) HMW DNA with 10 μl 10×Sau3AI buffer (NEB) and preheat at 37°C for 5 minutes. Then, 20 μl (2 units) of Sau3AI was added to the DNA solution, mixed slightly with a wide-bore pointed pipette, and incubated at 37°C. Aliquots of 15 μl were removed at 0, 5, 10, 20, 30 and 70 minutes and immediately mixed with 5 μl of 0.5M EDTA (pH 8.0) on ice to terminate the reaction. Samples were then analyzed by electrophoresis in a 0.3% agarose/TBE gel at 2 V/cm gel length in a cryogenic chamber.

The pilot experiment was repeated using an optimal incubation period of 20 minutes at 37°C to obtain large-scale partial digestions of DNA. (B) Size selection

Partially digested DNA was isolated by gradient centrifugation in a 5 to 40% sucrose gradient at 26,000 rpm for 13 hours at 20°C in a SW27 rotor. 0.8 ml aliquots (20 ml total) were carefully collected by inserting the capillary into the bottom of the centrifuge tube and pipetting up the gradient solution at a very slow rate. 20 μl of each sample was electrophoresed on 0.3% agarose gel at 2V/cm gel length for 36 hours. DNA fractions of 35 to 50 kb were pooled together. Dilute the sucrose solution with an equal volume of water, then precipitate DNA with 2 volumes of ethanol. Incomplete reactions were performed using standard methods. Ligation, packaging and transfection

Cosmid vector pHC80 was provided by Dr. Scot Hulbert. The vector was ligated to the insert DNA at a 2:1 molar ratio with a final concentration of 0.8 μg/μl. Ligation reactions were performed overnight at 16°C using 600 units of T4 DNA ligase (NEB, USA). The ligated DNA was packaged in vitro with Gigapack II packaging extract (Stratagene, USA) according to Stratagene's instructions, and then transfected into competent cells Escherichia coli NM554. Library screening

61440 cosmid colonies (greater than 5 genome equivalents) from 160 384-well microtiter plates were transferred to Hybond N+ filters (Amersham, USA) at two densities. In the first method, cosmid clones were replicated at low density (1536 colonies/11.5 x 15 cm filter) using a manual replicator (Genetix, UK) and grown overnight on LB/agar containing 10 μg/ml ampicillin. Cover the entire cosmid library with 40 filters. In the second method, cosmids were replicated at high density in rows using a Beckman Biomek ^™ motorized manipulator and grown using the same method as above. Using 3 x 3 rows, 3456 colonies were transferred to an 8.5 x 12 cm filter. To accurately localize positive colonies on a negative background, a reference cosmid colony (containing the RG103 marker gene) was grown at the first position of each 3×3 grid. The next 8 positions were grown with colonies from 8 microtiter plates of the cosmid library. At this point, 20 filters of 8.5×12 cm can cover the entire library. To hybridize with a specific probe, RG103 is mixed with the specific probe at a ratio of 1:4 to form a reference pattern.

Bacteria on the filter membrane were lysed and immobilized using the water steam bath method modified as follows: put the colony face up on two sheets of 3MM Whatman immersed in the lysis solution (0.5M NaOH, 1.5M NaCl), place it at room temperature for 4 minutes, pack The plastic box with the filter membrane was incubated in a steam bath at 85°C for 6 minutes, and then the filter membrane was transferred to a 3MM Whatman immersed in a neutralizing solution (1M Tris-HCl (pH7.4), 1.5M Nacl), Leave for 4 minutes. Dip in proteinase K solution (50mM Tris-HCl (pH8.0), 50mM EDTA (pH8.0), 100MmNaCl, 1% (v/v) sodium lauryl sarcosine, 250μg/μl proteinase K) in Incubate at 37°C for 20 minutes to remove protein and cellular debris. The filter membrane was gently washed with 2×SSC solution for 5 minutes at room temperature, and after drying, it was treated with UV at a distance of 10 cm for 2.5 minutes.

Hybridization was performed according to the following standard protocol: filters were treated with prehybridization solution (7% SDS, _{0.5M Na2PO4} (pH 7.2), 1 mM EDTA, 100 μg/ml ssDNA) at 65°C for 2 hours to overnight. Probes were labeled by random primer extension and hybridized overnight at 65°C with shaking. The filters were washed slightly (40 mM Na ₂ PO ₄ (pH 7.2), 1% SDS) at room temperature, and then incubated in the same solution at 65° C. for 20 minutes with gentle shaking. B. Results

The cosmid library was screened with three Xa21-linked markers (RG103, RAPD248 and RAPD818). Genomic Southern analysis showed that the copy numbers of the three markers in the resistant line were 8, 1 and 2, respectively (unpublished results). Six positive cosmid clones hybridizing to the RG103 marker were identified and confirmed by further Southern analysis. However, no positive clones containing RAPD248 and RAPD818 were identified.

Example 2

Identification of Xa21 gene

The 5 cosmid clones and 1 BAC clone isolated in Example 1 were further identified by restriction enzyme mapping. Figures 2A to 2E are partial restriction maps of cosmid clones. Figure 2F is a partial restriction map of a BAC clone.

An open reading frame (SEQ. ID. No. 1) was identified in one of the clones, pB806. It includes a promoter region, a putative intron and a portion of the 3' sequence. SEQ.ID.No.2 shows the deduced amino acid sequence. Putative introns have been excised.

The deduced amino acid sequence revealed two properties of the protein that indicate that it is encoded by a new class of plant disease resistance genes, referred to herein as RRK genes. First, the extracellular domains of the proteins encoded by these genes contain a block of approximately 23 tandem leucine-rich repeats (LRRs) with an average length of 24 amino acids. LRR motifs have been implicated in protein-protein reactions and ligand binding in a variety of proteins. The extracellular domain also contains a region between the LRRs and the signal peptide that contains the SWNTS motif, which is conserved in many proteins, including Df-9, PGIP, and RLK5. In addition, the protein contains a region that interacts with receptor-like protein kinases (PLPK) such as PLPK5 and TMK1 (Walker et al., Plant J. 3: 451 (1993); Chang et al. Plant Cell (Plant Cell ) 4: 1263 (1992); Valon et al., Plant Molecular Biology Plant Molec.Biol.) 23: 415 (1993)) and the tomato resistance gene product Pto (Martin et al., Science (Scince) 262: 1432 (1993) )) have a high degree of sequence identity. The signaling domain, extracellular domain (comprising LRR regions), transmembrane domain and cytoplasmic kinase domain are identified in SEQ.ID.No.2.

Figure 3 is a restriction map of another clone, pB822, used for the plasmid used in the transformation experiments described in Example 3 below. The Xa21 gene in this clone was also sequenced (SEQ. ID. No. 3). The deduced amino acid sequence (SEQ.ID.No.4) shows the same motif identified in SEQ.ID.No.2.

The protein kinase domain carries 11 subdomains, which contain 15 conserved protein kinase characteristic residues, flanked by a 33aa membrane-binding domain (aa 677-707) and a C-terminal domain. The putative intron is between two highly conserved residues P and E (aa892 and aa893) in the putative catalytic domain. Consensus sequences in subdomains VI (DIKSSN) and VIII (GTGYAAPE) clearly indicate that Xa21 has serine/threonine kinase (as opposed to tyrosine) activity.

A previous study demonstrated that phosphorylated RLK5 protein reacts with the kinase domain (KID) of a type 2C serine-threonine protein phosphatase (Stone et al., Science, 266:793-795 (1994)). KID binds phosphorylated LRRs containing RLK5 and TMK1 proteins, but not the S-associated receptor kinases ZmpK1 and RLK4. This result indicates that the Arabidopsis KID is functionally similar to the SH2 domain of animal proteins. Sequence alignment of Arabidopsis receptor-like kinases RLK5 and TMK1 with Xa21 reveals a group of conserved amino acids (N/Q)X(L/V)S(G/S)(L/A) around a serine residue (F/V)(P/E), the serine residue that is carboxy-terminal to the last residue (arginine), is highly conserved among all protein kinases (position 999 of the Xa21 gene product). The carboxy-terminal position shared by these proteins is similar to that of the carboxy-terminal phosphotyrosine in the oncogene product pp60 c-Src of Rous sarcoma virus, which is required for binding to SH2 domain-containing proteins. These conserved amino acids are absent in the S-associated receptor kinases ZmpK1, RLK4 and SRK6 and intercellular kinases that do not bind KIDs. Therefore, this region acts as a high-affinity and specific binding site for the KID protein. Therefore, the affinity for KID protein can be changed by changing the amino acid sequence of the Xa21 region, thereby regulating the intercellular signal transmission caused by the ligand binding of the LRR domain.

Example 3

Transformation of plants with the Xa21 gene

Rice plants can be transformed with the above-mentioned Xa21 gene to demonstrate that the gene confers resistance to Xanthomonas in susceptible plants. The method of Li et al., Plant Cell Rep. 12:250-255 (1993), was modified to introduce the gene into susceptible rice plants. Briefly, co-transformation was performed with the hygromycin construct pMON410 (supplied by Monsanto) and the Bluscreipt vector containing the sequence of interest. In addition, the Kpn fragment of pB822 was cloned into the pTA818 vector derived from the Invitrogen vector pcr1000 and containing the 1 kb fragment RAPD818 (Ronald et al., supra). The resulting plasmid was called pC822. Plants were selected with hygromycin (30 mg/L) and then screened for resistance to Xoo species 6.

Transformants were tested for Xanthomonas resistance using standard methods. Experiments were performed according to the method of Kaufma et al., Plant Disease Rep. 57:537-541 (1973). Briefly, Xoo species 6 were grown on PSA plates for 3 days. Scrape the bacteria, suspend them in water, and adjust the OD value to 10 ⁹ colony forming units/ml. The leaves of transformed plants (4 months after bombardment) were cut by dipping the scissors once in the suspension at a distance of 5 cm from the leaf tip. The plants were rated for the appearance of lesions 11 days after inoculation.

Figure 4 shows lesion length data from experiments performed using expression vectors containing genes from the pC822 clone. Individuals 106, -9, -22, 11, -17, -1, -12, -4, 16, and 29 developed from different transformants carried the pC822 construct and, compared to susceptible untransformed controls (IR24 ) compared with rice plants transformed with the vector (1-15) showed increased resistance.

Example 4

Isolation of RRK gene from tomato

As mentioned above, the Xa21 sequence can be used to isolate RRK genes from other plant species using degenerate primers or low stringency hybridization methods. This example illustrates the isolation and identification of two RRK genes from tomato using the degenerate primer method.

Degenerate primers were prepared such that the PCR (polymerase chain reaction) product amplified the sequence between the LRR and the kinase domain, thereby spanning the transmembrane domain. The forward primer was derived from a conserved motif in the LRR region of Xa21 and several other plant proteins (eg, cf-9, RLK5 and PGIP). Reverse primers were derived from conserved motifs from the Xa21 kinase domain and other plant serine-threonine kinase domains (e.g., RLK5, Pto, and Fen) (Martin et al., Plant Cell 6:1543-1551 (1994)) .

The degenerate primers used for PCR product amplification are as follows: 1.LRR region

TCA AGC AAC AAT TTG TCA GGN CA(A/G)AT(A/C/T)CC(SEQ.ID.No.5) 2. Kinase region

TAA CAG CAC ATT GCT TGA TTT NAN(G/A)TC NCG(G/A)TG(SEQ.ID.No.6)

TAA CAG CAC ATT GCT TGA TTT NAN(G/A)TC(G/A)CA(G/A)TG(SEQ.ID.No.7)

TAA CAG CAC ATT GCT TGA TTT NAN(G/A)TC(T/C)CT(G/A)TG(SEQ.ID.No.8)

PCR

The PCR conditions are as follows (20 μl reaction):

First loop:

94°C, 30 seconds (denaturation)

55℃, 30 seconds (annealing)

72°C, 1 minute (extended)

In the following 19 cycles, the annealing temperature was decreased by 1°C in each cycle. After the 20th cycle, the reaction solution was incubated at 72°C for 10 minutes.

After the primary amplification using the above primers, a secondary amplification was performed using the following specific primers:

TAAGCAACAATTTG(SEQ.ID.No.9)

       TAACAGCACATTGCTTGA(SEQ.ID.No.10)

The conditions for this expansion are as follows:

94°C, 15 seconds

55°C, 15 seconds

72°C, 15 seconds

After 35 cycles, the reaction was incubated at 72°C for 10 minutes.

The PCR product was cloned and used as a probe for screening the tomato cDNA library. Tomato cDNA was hybridized with oligo dT primers, ligated with EcoRI adapters, and cloned into the λGT11 vector to construct a library.

The above primers were used to isolate two tomato PCR products and cDNAs belonging to the RRK family of disease resistance genes. The first clone, TRK1 (Tomato Receptor Kinase 1), was a 250 bp PCR product and was used to isolate a partial cDNA. The DNA sequence is shown in SEQ.ID.No.11. The deduced amino acid sequence of TRK1 is shown in SEQ.ID.No.12.

This clone has one or two copies in the tomato genome, one of which is located on the short arm of chromosome 1 on the restriction map, next to the Xanthomonas campestris pv. vesicatoria resistance gene (Rxl) (Zu et al., Genetics 141:675-682 (1995)) (see Figure 5).

The second clone TRL1 (tomato receptor-like 1) is a 496bp PCR product. The DNA sequence is shown in SEQ.ID.No.13. The deduced amino acid sequence is SEQ.ID.No.14. TRL1 is within a few cM of the mutant mcn on chromosome 3 on the restriction map, and the mutant mcn causes punctate necrosis of the plant, which is a typical defensive phenotype.

The above results show that the Xa21 gene can be used to isolate RRK genes from other plant species. For example, the isolated TRK1 and TRL1 genes are important components of signaling pathways in plants that elicit defense responses. These genes can be used to engineer disease resistance in tomato and other plant species.

The above examples are intended to illustrate the invention and not to limit its scope. Other modifications of the invention will be apparent to those skilled in the art, and these are within the scope of the appended claims. All publications, plasmids and patent publications cited herein are incorporated by reference.

SEQ.ID.No.1

                                  RRK-FaagctttctaaattatttaactctaagtctgttattatccccaagtacatcatcatcatacataatatttcatattcacgacatccttaagctagatgcttttggccattctcttatctttttaaagaaattctctcccaattaagatgagagtgtcttctagcaatttgccagtttttacaatgtctttgagtcctcacacattttcatgatgttaccaataaattacggacgccgtgtttagttctaaagtttttcttcaaacttacaacttttcaatcgcatcaaaactttctcctacacacacaaactttcaacttttccatcacatcgttccaatttcaaccaaacttccaattttggtatgaactaaacacagccgaaaacaaaatctgtgtgttatggccctgtttagattctaacttttccattacatcaaactttcctacatacacgaactttcaacttttccgtcacatcgtttcaattttttaaaacttccatttttaacgtggaactaaacacaacctatataacggaatttgtcaaaaactcaatggtgaaagtcacacctcacaggaagggcgcgctctagtcaagacatcattaaacaggtacacaggttgtactagcttgtcatgtttatcttgcgtctgcgagacgtaaatccatgccaaacaaaagtgcttctatagagatatcataaggatatggtttggggccatatccaactgctcaggagagatctcgttcggaggtgaggttagatgttcacctctccacacataacgaaggcgatcttcttcgcatatgattaggcattagataaaataaccttaaaaaataaatcaatatgatttttttagaaaaaaattatatacactaagtataagcattgtcaaggaggaagaaacacacactcccatatagagagatagaaacatagctataggtagtgtcactgagtattttccatcacgcatatccatataaaattagggggtgttacatccataggtgtaaagttttggcatgttatatcgagtattacgtagaatgccgtattaggtgtccgggcactaataaaaaaataattacagaatccgttagtaaaccgcgagataaatttattaagcctaattaatcccatcattaacaaatgtttaccgtagcaccacattgtcaaatcatggagcaattaggtttaaaagattcgtctcgcaaattagtcataatctgtgcaattagttatttttagactatatttaagacttcgtacaggtgttcaaacgttcgatgtgacatggtgcaaaattttagggtgtcatctagacactcccttaattagaaagttaggaagaggcggtaaagaacgcagcatgactgaaactttgaaaatttgataaggtacaccaactggagtatcttttattttcattgaagactttgaccagaagagcttgacccgtttttcttggagtagccagtaatgtttcattcttttccttttgctgggacttctttttattttttttgacaggagccatttgttgggacttgggatccctttactgttataggaccagtgcttgaatccaaacactgcattgatcagctcagctcattgtagcgcactcctccgcatgcATGGCGAGATCACCAACGTCGGTCATGATCTCTTCTTTGCTGCTGCTGCTGTTGATCGGCCCAGCGAGCAGTGACGATGATGCTGCTGCTGCTGCTGCTCGTACCAGTACAGGCGGCGTCGCGGCGACGAACTCGCGCTGCTCTCTTTCAAGTCATCCCTGCTACACCAGGGGGGCTTGTACGCTGGCATCTTGGAACACGTCCGGCCACGGCCAGCACTGCACATGGGTGGGTGTTGTGTGCGGCCGCGCGCGCCGGCACCCACACAGGGTGGTGAAGCTGCTGCTGCGCTCGTCCAACCTGTCCGGGATCATCTCGCCGTCGCTGGGCAACCTGTCCTTCCTCAGGGAGCTGGACCTCAGCGACAACTACCTCTCCGGCGAGATACCACCGGAGCTCAGCCGTCTCAGCAGGCTTCAGCTGCTGGAGCTGAGCGGTAACTCCATCCAAGGGAGCATCCACGCGGCCATTGGAGCATGCACCAAGTTGACATCGCTAGACCTCAGCCACAACCAACTGAGATTGGTGCCAGCTGAAACATCTCTCGAATTTGTACCTTCACACCAATGGTTATGTCAGGAGAGATTCCATCTGATTTTGGGCAATCTCACTACGCCTTCAGTATTTGATTTGACCTGCAACAGATTATCACGGAGCTATACCTTCATCGCTAGGGCAGCTCAGCAGCAGTCTATTGACTATGAATTTTGTGCTACGAACAATCTAACTGGCATGATCCCCAATTCTATCTGGAACCTTTCGTCTCTAGCAGCGTTTAGCTGTCAAGCGAAAAACAAGCTAGGTGGTATGATCCCTACAAATGCATTCAAAACCCTTCACCTCCTCGAGGTGGTAGATATGGGCACTAACCGATTCCATGGCAAAATCCCTGCCTCAGTTGCTAATGCTTCTCATCTGACACGGCTTCAGATTGATGGCAACTTGTTCAGTGGAATTATCACCTCGGGGTTTGGAAGGTTAAGAAATCTCACAACACTGTATCTCTGGAGAAATTTGTTTCAAACTAGAGAACAAGAAGATTGGGGGTTCATTTCTGACCTAACAAATTGCTCCAAATTACAAACATTGGACTTGGGAGAAAATAACCTGGGGGGAGTTCTTCCTAATTCGTTTTCCAATCTTTCCACTTCGCTTAGTTTTCTTGCACTTGATTTGAATAAGATCACAGGAAGCATTCCAAAGGATATTGGCAATCTTATTGGCTTACAACATCTCTATCTCTGCAACAACAATTTCAGAGGGTCACTTCCATCATCGTTGGGCAGGCTTAGAAACTTAGGCATTCTAGTCGCCTACGAAAACAACTTGAGCGGTTCGATCCCATTGGCCATAGGAAATCTTACTGAACTTAATATCTTACTGCTCGGCACCAACAAATTCAGTGGTTGGATACCATACACACTCTCAAACCTCACAAACTTGTTGTCATTAGGCCTCTCGCACCTCGCACCACAATCAGGGTTGGATACCTACACATCTCAACCTCACAACTGTGTCATAGCCTTCACTATACCTAGTGGGTCCCAAATACCCCAGGTGAAATTAATTCAAATAGTCCAAACACCTATCAAAAAGATGATCAATGTATCAAAAAATACACTTGGAGGGATCAGATACCCACAAGAAATAGGGCATCTCAAAAATCTAGTAGAATTCATGCAGAATCGAATAGATATCAGTAAAATCCCTAACACGCTTGGTGATTGCCAGCTCTTACGGTATCTTTATCTGCAAAATAATTTGTTATCTGGTAGCATCCCATCAGCCTTGGGTCAGCTGAAAGGTCTCGAAACTCTTGATCTCTCAAGCAACAATTTGTCAGGCCAGATACCCACATCCCTTAGCAGATATTACTATGCTTCATTCCTTGAACCTTTCTTTCAACAGCTTTGTGGGGGAAGTGCCAACCATTGCGTGCTTTCGCAGATGCATCCGGGATCTCAATCCAAGGCAATGCCAAACTCTGTGGTGGAATACCTGATCTACATCTGCCTCGATGTTGTCCCATTACTAGAGAACAGAAAGCATTTTCCAGCTCTACCTATTTCTGTTTCTCTGGTCGCAGCACTGGCCATCCTCTCATCACTCTACTTGCTTATAACCTGGAACAAGAGAACTAAAAAGGGAGCCCCTTCAAGAACTTCCATGAAAGGCCACCCATTGGTCTCTTATCCGCAGTTGGTAAAAGCAACAGATGGTTTCGCGCCGACCAATTTGTTGGGTTCTGGATCATTTGCCTCAGTATACAAACGAAAGCTTGAAAATCCTAAGGCACTCAAGAGTTTCACTGCCGAATGTGAAGCACTACGAAATATGCGACATCGAAATCTTGTCAAGATAGTTACAATTTGCTCGAGCATTGATAACAGAGGGAACGATTTCAAAGCAATTGTGTATGACTTCATGCCCAACGGCAGTCTGGAAGATTGGATACACCCTGAAACAAATGATCAAGCAGACCAGAGGCACTTGAATCTGCATCGAAGAGTGACCATACTACTTGATGTTGCCTGTGCATTGGACTATCTTCACCGCCATGGCCCTGAACCTGTTGTACACTGTGATGTTAAATCAAGCAATGTGCTGTTAGATTCTGATATGGTAGCGCATGTTGGAGATTCTGGGCTTGCAAGAATACTTGTTGATGGGACCTCATTGATACAACAGTCAACAAGCTCGATGGGATTTAGAGGGACAATTGGCTATGCAGCACCAggtcagcaagtccttccagtattttgcattttctgatctctagtgctatatgaaatagtttttacctctagtgaaactgatggagaatataagtaattaattgaactaattaaattgcacaaaaataagattatttgccatatctattcagatgctaaatatagctagttcatagaggtacatattttttttatataggaatctagagctactacacactcaaatcaaattatgggtgttttctgctctacactgcaatatgaaatgattatcagaaggatcaaatttgagtaaatttgtcaattctacatttaagaaacacttttttttgtatgtactagttattacaattttttatttcaagaacttgcattgaccatgaaaagtacttggtactacttctaattcccacatggaggtggtgaaaataatatagatacaaaaacgaagtatcatatgttgtgtgatatactataatcacaatgaacacaaacaggattcgtacaaaagtaattggccatcatagcaactgattgcttggggtaactgtatagcacaatcataccaaatttctttagatatgtatttgtaaattagattcttaaagttaaatatgaaatttcattggtatttatgtttctttatataataaaaattaatccaacctttacatctaccatttgtccagccatccttgttatttgtgatatttaacacgtaattttacataattatacatccaagttctttttatttaacactggaaatttgaaatcgtatttcctactcaaacaGAGTATGGCGTCGGGCACATTGCATCAACACATGGAGATATTTACAGCTATGGAATTCTAGTGCTGGAAATAGTAACCGGGAAGCGGCCAACTGACAGTACATTCAGACCCGATTTGGGCCTCCGTCAGTACGTTGAACTGGGCCTACATGGCAGAGTGACGGATGTTGTTGACACGAAGCTCATTTTGGATTCTGAGAACTGGCTGAACAGTACAAATAATTCTCCATGTAGAAGAATCACTGAATGCATTGTTTCGCTGCTTAGACTTGGGTTGTCTTGCTCTCAGGATTTNCCATTGAGTAGACGCCACCCGGAGATATCACCGACGAACTGAatgccatcaaacagaatctctccggagttgtttccagtgtgtgaaggtgcgagcctcgaattctgatgttatgtcttgtaatgttttattgccactagtcttcagattggaatgctcttccgatcagacttcttcagtggtatctaccacacgatcactaaagtcatcgtggctatttcctgatccagcatatctgatcatgcatgttctgtgttttatacctgtattttactctgaattgccacacctcaaccctgcctctgtttgtttggcatacaaaagatagtgatgagtatattgtttcaggggcttcctagttggcgtgtgtgcttaccggcacgcacgcagcccgagggtgggtttctttttttttccattgttattccgttgcttttttccaccacggtagattttttttttctggatttccattttttccgttgtttttctctatcgcttatgctggcggatttttttccgtggtttttttttcaagacgagtatatctaatgtaactaacatgttacttttagataacgatggttattaagataagatttttttctggaagatttttgtaagtaaatggtaaaaaatatggaaatggaaacggaaatagttttgctgttataccgatcgtttccatatttaccgtattcttatagaaattaccgtntcttataatatggtaattaccgtatttctaaatatgttgatatcgattttgctatatatttgtcgacSEQ.ID.No.2

RRK-FMARSPTSVMISSLLLLLLIGPASSDDDAAAAAARTSTGGVAATNSRCSLSSHPCYTRGACTLASWNTSGHGQHCTWVGVVCGRARRHPHRVVK

LLLRSS N LSGIISPSLGNLSFLRELDLSD NYLSGEIPPELSRLSRLQLLELSG NSIQGSIHAAIGACTKLTSLDLSH NQLRLVPAETSLEFVPSHQWLCQERFHLILGNLTTPSVFDLTC NRLSRSYTFIARAAQQQSIDYEFCAT NNLTGMIPNSIWNLSSLAAFSCQAKNKLGGMIPTNAFKTLHLLEVVDMGT NRFHGKIPASVANASHLTRLQIDG NLFSGIITSGFGRLRNLTTLYLWR NLFQTREQEDWGFISDLTNCSKLQTLDLGE NNLGGVLPNSFSNLSTSLSFLALDL NKITGSIPKDIGNLIGLQHLYLCN NNFRGSLPSSLGRLRNLGILVAYE NNLSGSIPLAIGNLTELNILLLGT NKFSGWIPYTLSNLTNLLSLGLSHLAPQSGLDTYTSQPHNCVIAFTIPS GSQIPQVKLIQIVQTPIKKMINVSK NTLGG IRYPQEIGHLKNLVEFMQNRID ISK IPNTLGDCQLLRYLYLQN NLLSGSIPSALGQLKGLETLDLSS NNLSGQIPTSLSRYYYASFLEPFFQQLCGGSANHCVLSQMHPGSQSKAMPNSVVEYLIYICLDVVPLLENRKHFPALPISVSLVAALAILSSLYLLITWNKRTKKGAPSRTSMKGHPLVSYPQLVKATDGFAPTNLLGSGSFASVYKRKLENPKALKSFTAECEALRNMRHRNLVKIVTICSSIDNRGNDFKAIVYDFMPNGSLEDWIHPETNDQADQRHLNLHRRVTILLDVACALDYLHRHGPEPVVHCDVKSSNVLLDSDMVAHVGDSGLARILVDGTSLIQQSTSSMGFRGTIGYAAPEYGVGHIASTHGDIYSYGILVLEIVTGKRPTDSTFRPDLGLRQYVELGLHGRVTDVVDTKLILDSENWLNSTNNSPCRRITECIVSLLRLGLSCSQDL ^* (F)PLSRRHPEISPTNZ ^* L：“N”＝“G”F：“N”＝“C”SEQ.ID .No.3: ORF 3918 (3075bp interrupted by an intron (843bp)).ID.No.41025 amino acids A MISLPLLLFVLLFSALLLCPSSS 23B DDDGDAAGDELALLSFKSSLLYQGGQSLASWN 55

TSGHGQHCTWVGVVCGRRRRRHPHR 80C VVK LLLRSSN LSGIISPS 98

LGNLSFLRE LDLGDNY LSGEIPPE 122

LSRLSRLQL LELSDNS IQGSIPAA 146

IGACTKLTS LDLSHNQ LRGMIPREI 171

GASLKHLSN LYLYKNG LSGEIPSA 195

LGNLTSLQE FDLSFNR LSGAIPSS 219

LGQLSSLLT MNLGQNN LSGMIPNS 243

IWNLSSLRA FSVRENK LGGMIPTNA 268

FKTLHLLEV IDMGTNR FHGKIPAS 292

VANASHLTV IQIYGNL FSGIITSG 316

FGRLRNLTE LYLWRNL FQTREQDDWGFISD 346

LTNCSKLQT LNLGENN LGGVLPNSF 371

SNLSTSLSF LALELNK ITGSIPKD 395

IGNLIGLQH LYLCNNN FRGSLPSS 419

LGRLKNLGI LLAYENN LSGSIPLA 443

IGNLTELNI LLLGTNK FSGWIPYT 467

LSNLTNLLS LGLSTNN LSGPIPSE 491

LFNIQTLSIMINVSKNN LEGSIPQE 516

IGHLKNLVE FHAESNR LSGKIPNT 540

LGDCQLLRY LYLQNNL LSGSIPSA 564

LGQLKGLET LDLSSNN LSGQIPTS 588

LADITMLHS LNLSFNS FVGEVPT 611

 IGAFAAASG ISIQGNAKLCGGIP             634D    DLHLPRCCPLLENRKH                     650E    FPVLPISVSLAAALAILSSLYLLITW           676F    HKRTKK                               682G    GAPSRTSMKGHPLVSYSQLVKATDG            707H    FAPTNLLGSGSFGSVYKGKLNIQDHVAVKVLKLENPKALKSFTA          751

ECEALRNMRHRNLVKIVTICSSIDNRGNDFKAIVYDFMPNGSLE 795

DWIHPETNDQADQRHLNLHRRVTILLDVACALDYLHRHGPEPVV 839

HCDIKSSNVLLDSDMVAHVGDFGLARILVDGTSLIQQSTS 879

SMGFIGTIGYAAPEYGVGLIASTHGDIYSYGILVLEI 916

VTGKRPTDSTFRPDLGLRQYVELGLHGRVTDVVDTKLILDSENW 960

LNSTNNSPCRRITECIVWLLRLGLSCSQELPSSRTPTGDIIDEL          1004I    NAIKQNLSGLFPVCEGGSLEF                                 1025SEQ.ID.No.5TCAAGCAACAATTTGTCAGGNCA A/G AT A/C/T CCSEQ.ID.No.6TAACAGCACATTGCTTGATTTNAN G/A TCNCG G/A TGSEQ.ID.No.7TAA CAG CAC ATT GCT TGA TTT NAN (G/ A)TC (G/A)CA (G/A)TGSEQ.ID.No.8TAA CAG CAC ATT GCT TGA TTT NAN (G/A)TC (T/C)CT (G/A)TGSEQ.ID.No .9TAAGCAACAATTTGSEQ.ID.No.10TAACAGCACATTGCTTGASEQ.ID.No.11TRK1开放读码框(DNA序列).ID.No..ID.No.13TRL1 DNA序列TCAAGCAACAATTTRTCAGGACAAATACCTTCAGGCTTGGCCAATGTGACCACACTGGCAGCATTTAACGTTTCTTTCAATAATCTGTCTGGGCCACTGCCTCTTAACAAAGATTTGATGAAGTGTAATAGTGTTCAGGGAAACCCCTTTCTGCAATCGTGCCATGTATTTTCTCTATCAACACCTTCTACAGATCAGCAGGGAAGAATAGGGGACTCACAAGATTCTGCTGCGTCTCCTTCAGGTTCAACCCAGAAAGGAGGGTGCAGCGGTTTCAACTCCATAGAGATTGCATCCATAACATCTGCGGCAGCTATTGTGTCAGTTCTTCTTGCTCTGATAGTCCTGTTCTTTTACACCAGAAAATGGAATCCAAGATCTAGAGTTGCTGGATCTACCAGGAAAGAAGTCACAGTGTTTACAGAAGTTCCGGTTCCTTTAACATTTGAAAATGTAGTGCGGGCCACAGAGATCTCAAATCAAGCAATGTGCTGTT

Claims (13)

1. An isolated nucleic acid construct, characterized in that it contains the polynucleotide sequence of the disease resistance gene, the polynucleotide contains the Xa21 gene, and the described Xa21 gene encodes SEQ.ID.No. 2 or the Xa21 polypeptide shown in SEQ.ID.No.4. 2. The nucleic acid construct of claim 1, wherein the polynucleotide sequence is a full-length gene. 3. The nucleic acid construct according to claim 1, wherein said polynucleotide sequence comprises the open reading frame of SEQ.ID.No.1 or SEQ.ID.No.3. 4. The nucleic acid construct according to claim 1, further comprising a promoter operably linked to the polynucleotide sequence of the disease resistance gene. 5. The nucleic acid construct of claim 4, wherein the promoter is a tissue-specific promoter. 6. The nucleic acid construct of claim 4, wherein the promoter is a constitutive promoter. 7. A transgenic plant cell, characterized in that it contains a recombinant expression cassette containing a plant promoter operably linked to the Xa21 polynucleotide sequence of claim 1. 8. The plant cell of claim 7, wherein the plant promoter is a heterologous promoter. 9. The plant cell of claim 7, wherein the plant cell is a rice plant cell. 10. A method for increasing plant resistance to Xanthomonas, characterized in that the method comprises introducing a recombinant expression cassette into the plant, the expression cassette containing the disease resistance gene operably linked to claim 1 Plant promoters of polynucleotide sequences. 11. The method of claim 10, wherein the plant tissue is from rice. 13. The method of claim 10, wherein the promoter is a tissue-specific promoter. 14. The method of claim 10, wherein the promoter is a constitutive promoter.

Images